Figure 4.

Common peptide fragmentation methods. Peptides are cleaved along the peptide backbone to sequence the peptide. CID/CAD entails acceleration of the kinetic energy of ions to promote energetic collisions with a target gas, thus causing conversion of the ion’s kinetic energy to internal energy and ultimately resulting in ion fragmentation. Most fragmentation pathways rely on proton transfer. For trapping instruments that employ resonant excitation, the waveform used to accelerate a precursor ion is specific for a particular m/z value, thus only the selected precursor ion is activated. The resulting fragment ions are not excited and thus do not dissociate further, allowing detection of an array of predominantly both b and y ions (as well as others). In contrast to resonant excitation in trapping instruments, in beam-type CID both the selected precursor ions and any resulting fragment ions are passed through a collision region which enables further activation and dissociation of the fragment ions. This means that the less stable b ions will frequently decompose to very small ions. Thus, both peptide precursors and their fragment ions are activated and mostly y-type fragment ions persist. Unlike CID, ECD and ETD fragmentation relies on the gas phase reaction of the peptide ions with a thermal electron or an ETD reagent, respectively. The electron transfer-driven fragmentation mechanisms create mostly c- and z-type ions.